Chromatin Remodeling of the Epidermal Differentiation Complex

 描述(申请人提供)：皮肤表面的表皮为生物体提供了第一道防线。表皮分化复合体(EDC)基因的表达对这种表皮屏障功能至关重要。EDC编码4个基因家族(Flg-like、LCE、SPRR和S100)，它们是协同表达的，提供了定义功能性表皮角质形成细胞的基本细胞成分。EDC的发现距今已有20年，角质形成细胞中EDC伴随激活的分子机制一直没有得到解决。使用一种前所未有的、全面的皮肤生物学基因组学方法，我们直接重新审视这个重要的问题。增强子是基因组中重要的非编码调控元件。增强子通过转录因子结合介导的与相应基因启动子的近端环路来激活基因表达。我们最近的研究在EDC中发现了一个高度保守的增强子，即923，它的调节活性与小鼠发育中的表皮分化和EDC转录的空间和时间模式一致，并具有特异性。利用创新的染色体构象捕捉方法，我们进一步确定了角质形成细胞分化过程中EDC相对于923增强子的动态染色质重塑。在AP-1转录因子与923失去结合后，EDC染色质的异常重塑和抑制的EDC基因激活进一步支持了923增强子活性对EDC激活的作用。这导致我们提出了以下假设。923增强子是一个关键的调控增强子，它强加了染色质结构的特定配置，以协调EDC的转录激活，从而促进表皮分化。我们提出了以下目标：目的1.确定EDC基因在小鼠和人类中协调表达的保守表观遗传学机制。在这里，我们将检验这样的假设，即923从CTCF结合的绝缘体中释放，从而允许可访问的AP-1结合，并且923增强激活的EDC基因的表达。我们将使用基因组学方法(4C-seq)来定义与923、微丝蛋白和已知的CTCF结合位点有关的染色质相互作用，并确定控制这些相互作用的表观遗传机制(平行的芯片-seq、RNA-seq和ATAC-seq研究)。目的2.测定923增强子在表皮发育过程中的缺失情况。我们使用最先进的CRISPR/Cas9介导的基因组工程方法，成功地产生了5个独立的923只缺失(3)和连系(2)的小鼠。将进行分子和细胞研究以确定小鼠的表型。这些目标的实现将对我们理解染色质结构与基因表达的分子基础产生重大影响，其分辨率在皮肤生物学中达到前所未有的水平，并与所有人类组织类型相关。这项研究还将提供必要的工具，以开发新的策略，以纠正异常表皮分化的靶向性表观遗传和染色质重塑疗法在医学上。

 DESCRIPTION (provided by applicant): The epidermis at the surface of the skin provides the first line of defense for an organism. The expression of the Epidermal Differentiation Complex (EDC) genes is critical to this epidermal barrier function. The EDC encodes 4 gene families (FLG-like, LCE, SPRR, and S100) that are coordinately expressed and provide the essential cellular components that define a functional epidermal keratinocyte. It has been 20 years since the discovery of the EDC and the molecular mechanisms that govern concomitant activation of the EDC in the keratinocyte continues to elude the field. Using an unprecedented and comprehensive genomics approach to skin biology, we directly revisit this important question. Enhancers are important regulatory elements in the genome that are noncoding. Enhancers activate gene expression by proximal looping to the respective gene promoter that is mediated by transcription factor binding. Our recent studies identified a highly conserved enhancer within the EDC, namely 923, whose regulatory activity coincided with and is specific to the spatial and temporal patterning of epidermal differentiation and EDC transcription across the developing mouse epidermis. Using innovative chromosomal conformation capture methods, we further identified dynamic chromatin remodeling of the EDC with respect to the 923 enhancer during keratinocyte differentiation. A role for 923 enhancer activity for EDC activation was further supported by aberrant EDC chromatin remodeling and repressed EDC gene activation upon the loss of AP-1 transcription factor binding to 923. This led us to propose the following hypothesis. The 923 enhancer is a critical regulatory enhancer that imposes a specific configuration of the chromatin architecture for coordinate transcriptional activation of the EDC for epidermal differentiation. We propose the following aims: Aim 1. Identify the conserved epigenetic mechanisms of coordinate EDC gene expression in mice and humans. Here we will test the hypothesis that 923 is released from CTCF-bound insulators thereby allowing accessible AP-1 binding and for 923 to enhance activated EDC gene expression. We will use genomics approaches (4C-seq) to define the chromatin interactions with respect to 923, filaggrin, and a known CTCF binding site and identify the epigenetic mechanisms that govern these interactions (parallel ChIP-seq, RNA-seq, and ATAC-seq studies). Aim 2. Determine the loss of the 923 enhancer in epidermal development. We have successfully generated 5 independent lines of 923 deleted (3) and floxed mice (2) using the state-of-the-art CRISPR/Cas9-mediated genome engineering method. Molecular and cellular studies will be performed to determine the mouse phenotypes. Accomplishments of these aims will have a significant impact on our understanding of the molecular underpinnings linking chromatin architecture to gene expression at an unprecedented resolution in skin biology and relevant to all human tissue types. This study will also provide the necessary tools to develop novel strategies to correct aberrant epidermal differentiation with targeted epigenetic and chromatin remodeling therapies in medicine.